US5837812A - Human islet, human brain and mouse brain glutamic acid decarboxylase GAD polypeptides - Google Patents

Human islet, human brain and mouse brain glutamic acid decarboxylase GAD polypeptides Download PDF

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US5837812A
US5837812A US08/308,952 US30895294A US5837812A US 5837812 A US5837812 A US 5837812A US 30895294 A US30895294 A US 30895294A US 5837812 A US5837812 A US 5837812A
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Leonard Harrison
Margot Honeyman
David Cram
Henry De Aizpurua
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Walter and Eliza Hall Institute of Medical Research
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Amrad Corp Ltd
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to the identification, cloning and sequencing of nucleic acid molecules encoding an isoform of the enzyme glutamic acid decarboxylase and further relates to the use of these molecules and/or peptides and polypeptides encoded thereby in diagnostic tests for Insulin Dependent Diabetes Mellitus and other diseases in which glutamic acid decarboxylase is an autoantigen and in the treatment of patients suffering from these diseases.
  • GAD glutamic acid decarboxylase
  • GAD is co-precipitated with a separate 64K protein, the latter distinguished by tryptic products of M r 37,000/40,000 that are distinct from a M r 50,000 product of GAD.
  • GAD comprises at least two isoforms encoded by separate genes (16, 17, 18).
  • the predicted molecular weights of the known isoforms are approximately 67,000 and 65,000 (referred to as the "67K” and "65K” isoforms, respectively).
  • the distribution of GAD isoforms in different tissues in still not well defined, but it is likely that the 65K isoform accounts for the GAD component of the 64K autoantigen (17).
  • HB human brain
  • HI human pancreatic islet
  • MB mouse brain
  • recombinant GAD proteins corresponding to the 67K isoform and their fragments and derivatives were used as an antigen to detect antibodies and T-cells reactive with GAD thereby forming a basis for a new range of diagnostics and therapeutics for diseases of the type including preclinical and clinical IDDM and SMS and other diseases in which GAD is an autoantigen.
  • a first aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence which encodes or is complementary to a sequence which encodes human or mouse glutamic acid decarboxylase (GAD) corresponding to a 67K isoform of the enzyme or antigenically active fragments or derivatives thereof.
  • GAD glutamic acid decarboxylase
  • Another aspect of the present invention provides a synthetic peptide or polypeptide displaying the antigenicity of all or a portion of the 67K isoform of GAD or a fragment thereof and reactive with autoantibodies and/or T-cells.
  • Yet another aspect of the present invention contemplates a method for the detection of antibodies to GAD in a sample which method comprises contacting a peptide or polypeptide corresponding to all or an antigenic portion of the 67K isoform of GAD with said sample for a time and under conditions sufficient for a complex to form between the peptide or polypeptide and an antibody reactive to GAD and then detecting the complex.
  • Still yet another aspect of the present invention provides a method for detecting diseases of the type including IDDM and SMS, or for screening asymptomatic individuals, by the detection and/or determination of the titre of autoantibodies in a biological sample from said individual, said method comprising contacting said sample with a peptide or polypeptide corresponding to all or an antigenic portion of the 67K isoform of GAD for a time and under conditions sufficient to form a complex between the peptide or polypeptide and an antibody reactive to GAD and then detecting the complex and/or the amount of peptide or polypeptide which has been bound in a complex
  • the present invention also provides a method of reducing autoantibodies and/or autoreactive T-celis to GAD in a patient in need thereof and/or to desensitise or induce tolerance to eliminate or diminish reactivity of autoreactive T-cells or autoantibodies to the autoantigen, said method comprising administering to said patient GAD reactive T-cell lines or clones or cell membranes and/or receptors for the antigen from said GAD reactive T-cell lines or clones for a time and under conditions sufficient to act as immunogens to induce inhibition and/or reduction of T-cell responses to GAD autoantigen.
  • FIG. 1 shows a comparison of the DNA sequences corresponding to human brain GAD (HBGAD); SEQ ID NO.:1 and human islet GAD (HIGAD); SEQ ID NO.:2.
  • FIG. 2 shows the deduced amino acid sequences of HBGAD (SEQ ID NO.:3) and HIGAD (SEQ ID NO.:4) and their alignment with the equivalent region in the feline GAD (SEQ ID NO.:5) (amino acids 218-398).
  • FIG. 3 shows the nucleotide sequence and deduced amino acid sequence corresponding to the full length mouse brain GAD (MBGAD) (SEQ ID NO.:6).
  • FIG. 4 shows the nucleotide sequence and deduced amino acid sequence corresponding to the N-terminal fragment of MBGAD designated MBGAD12 (SEQ ID NO.:7) that encodes amino acids 1-204 of the published feline GAD sequence (10).
  • FIG. 5 shows the nucleotide sequence and deduced amino acid sequence corresponding to the mid-region fragment of MBGAD, designated MBGAD34 (SEQ ID NO.:8) corresponding to amino acids 198-404 of the published feline GAD sequence.
  • FIG. 6 shows the nucleotide sequence and deduced amino acid sequence corresponding to the C-terminal fragment of MBGAD, designated MBGAD56 (SEQ ID NO.:9) corresponding to amino acids 392-593 of the published feline GAD sequence.
  • FIG. 7 shows the full length nucleotide sequence and deduced amino acid sequence corresponding to human brain GAD (HBGAD-FL); (SEQ ID NO.:10).
  • FIG. 8 shows the nucleotide sequence and deduced amino acid sequence corresponding to the N-terminal fragment of HBGAD, designated HBGAD17 (SEQ ID NO.:11) corresponding to amino acids 1-250 of the published feline GAD sequence.
  • FIG. 9 shows the nucleotide sequence and deduced amino acid sequence corresponding to the mid region fragment of HBGAD or HIGAD, designated HBGAD14 or HIGAD14 (SEQ ID NO.:12) corresponding to amino acids 208-404 of the published feline GAD sequence.
  • FIG. 10 shows the nucleotide sequence and deduced amino acid sequence corresponding to the C-terminal region fragment of HBGAD, designated HBGAD65 (SEQ ID NO.:13), corresponding to amino acid 392-594 of the published feline GAD sequence.
  • FIG. 11 shows the full length nucleotide sequence and deduced amino acid sequence corresponding to human islet GAD (HIGAD-FL); (SEQ ID NO.:14).
  • the present invention provides a nucleic acid molecule comprising a nucleotide sequence which encodes or is complementary to a sequence which encodes human or mouse glutamic acid decarboxylase (GAD) corresponding to a 67K isoform of the enzyme or antigenically active fragments or derivatives thereof.
  • GAD glutamic acid decarboxylase
  • 67K isoform is meant the form of GAD having approximately M r 67,000 and/or any fragments, derivatives, homologues and/or immunological relatives thereof and which are distinguishable and/or otherwise distinct from the M r 65,000 form of GAD and which reacts preferentially to T-cells and/or autoantibodies from individuals with clinical or preclinical IDDM, SMS and/or other similar diseases.
  • the GAD is human pancreatic islet GAD (HIGAD), human brain cell GAD (HBGAD) and/or mouse brain cell GAD (MBGAD).
  • the nucleic acid molecule is DNA, at least a part of which has a nucleotide sequence substantially corresponding to the sequence shown in FIGS. 1, 3, 7 and/or 11 or a fragment, derivative, homologue or or immunological relative thereof or one or more sequences complementary thereto.
  • the present invention however also extends to any single or multiple nucleotide substitutions, deletions and/or additions to the sequence shown in FIGS.
  • the ribonucleotide sequence will, in a preferred embodiment, be substantially complementary to one or more of the sequences shown in FIGS. 1, 3, 7 and/or 11 or a fragment, derivative, or homolgue thereof.
  • the present invention also provides a recombinant nucleic acid (e.g. DNA) molecule comprising a nucleotide sequence as described above operably linked to an expression control sequence.
  • a recombinant molecule may, for example, comprise an expression vector.
  • the present invention further extends to a host cell such as a bacterium, yeast, mammalian or insect cell transformed with such a recombinant molecule.
  • a preferred mammalian cell line is the Chinese Hamster Ovary (CHO) cell line.
  • Another aspect of this invention is directed to a synthetic (e.g. recombinant) peptide or polypeptide displaying the antigenicity of all or a portion of an isoform of GAD which is reactive with autoantibodies and/or T-cells.
  • a synthetic (e.g. recombinant) peptide or polypeptide displaying the antigenicity of all or a portion of an isoform of GAD which is reactive with autoantibodies and/or T-cells.
  • Such a synthetic peptide or polypeptide may, for example, be prepared by recombinant means such as by the expression of a host cell transformed with the recombinant molecules described above.
  • the peptide or polypeptide may be fused to another peptide or polypeptide.
  • it may be prepared by chemical synthesis, such as by the well-known Merrifield solid-phase synthesis procedure.
  • the synthetic (eg. recombinant) peptide or polypeptide may or may not retain GAD enzymatic activity.
  • synthetic GAD or fragments thereof represent a preferred embodiment, the present invention also emends to biologically pure preparations of the naturally occurring enzyme or its fragrments.
  • biologically pure is meant a preparation of at least 60%, preferably at least 70%, more preferably at least 80% and still more preferably at least 90% by weight enzyme.
  • the present invention extends to naturally occurring or synthetic peptide or polypeptides corresponding to MBGAD, HIGAD and/or HBGAD and to nucleotide sequences coding for same as well as to fragments, derivatives, homolgoues or immunological relatives thereof.
  • fragments are shown in FIGS. 2, 4, 5, 6, 8, 9 and 10.
  • derivatives is meant to include any single or multiple amino acid substitution, deletion and/or addition relative to the naturally occurring sequence or to the sequence as shown in FIGS. 1, 3, 7 and/or 11 and including any single or multiple substitution, deletion and/or addition to other molecules associated with the peptide or polypeptide including carbohydrate lipid and/or other proteinacious moieties.
  • Such derivatives therefore, include glycosylated or non-glycosylated forms or molecules with altered glyclosylation patterns.
  • the present invention also contemplates a method for the detection of autoantibodies associated with IDDM which method comprises contacting a peptide or polypeptide corresponding to all, or an antigenic portion of, GAD, which GAD corresponds to the 67K isoform of the enzyme, or a fragment or derivative thereof with a biological sample from a patient to be tested for a time and under conditions sufficient for a complex to form between the peptide or polypeptide and an antibody reactive to GAD and then detecting the complex.
  • the biological sample is serum.
  • the peptide or polypeptide is immobilised onto a solid support before, during or after contact with the serum. Methods of detection are well known and include colorimetric, fluorometric and radioactive procedures.
  • detection means can also be used such as involving agglutination.
  • This assay can be varied in any number of ways without departing from the scope of the present invention.
  • the present invention also extends to the use of a peptide or polypeptide corresponding to the 67K isoform of GAD, or antigenic fragments thereof, as an antigen in a diagnostic test for diseases of the type including IDDM and SMS, or for screening asymptomatic individuals by detection or determination of the titre of antibodies in a patient's serum, for example using ELISA or RIA technology or an agglutination assay using antigen-coated beads or the like.
  • This aspect of the present invention may conveniently be carried out by the detection and/or determination of the titre of autoantibodies in a biological sample (e.g. serum) from a human subject, said method comprising contacting said sample with a peptide or polypeptide corresponding to an antigenic portion of the 67K isoform of GAD or a fragment or derivative thereof for a time and under conditions sufficient for a complex to form between the peptide or polypeptide and an antibody reactive to GAD and then detecting the complex and/or amount of peptide or polypeptide which has been bound in the complex.
  • the peptide or polypeptide is immobilised onto a solid support before, during or after contact with the sample and the peptide or polypeptide is as hereinbefore defined.
  • such diseases may be detected or at least a negative result re-confirmed or otherwise by screening for GAD associated immune complexes. It is possible, for example, that a negative autoantibody result could have been caused by autoantibodies forming complexes with GAD thereby not being available for binding in the aforementioned assay.
  • an anti-GAD antibody e.g. a monoclonal antibody
  • the anti-GAD antibody is first immobilised onto a solid support.
  • An anti-immunoglobulin antibody generally with a label or other reporter molecule attached, is then used to screen for the antibody component of the GAD complex.
  • the invention also extends to use of the peptides and/or polypeptides, or fragments, or derivatives of the present invention in the treatment of patients.
  • methods of treatment include their use as an adsorbent to remove autoantibodies or autoreactive cells from a patient, their use in direct administration to a patient as a means of desensitising or inducing tolerance to eliminate or diminish reactivity of autoreactive T-cells or autoantibodies to the IDDM autoantigen or to generate T-cell lines or clones to be used for or as therapeutic agents.
  • the method of treatment includes but is not limited to the following examples of treatment.
  • a first example of treatment is desensitisation or tolerance induction using an effective amount of GAD peptide or polypeptide or fragments thereof to alter T-cell recognition of GAD and induce T-cell suppression. This may be achieved by using the known effect of certain ultraviolet wavelengths, especially UV-B, to modify antigen presentation through the skin (see 19). Effective amounts of GAD peptide or polypeptide or fragments thereof would be applied epicutaneously to the skin of subjects exhibiting peripheral blood T-cell reactivity to GAD, after exposure of skin to UV-B radiation. Treatment would be repeated until such time that T-cell reactivity to GAD was suppressed.
  • a second treatment involves application of GAD to the skin together with one or more cytokines such as but not limited to TNF ⁇ or ⁇ .
  • a third treatment involves T-cell immunisation whereby T-cell lines are generated to GAD peptide or polypeptide or fragments thereof by standard procedures, calls attenuated by fixation with agents such as glutaraldehyde or paraformaldehyde, washed under sterile conditions and re-injected to patients for a time and under conditions causing suppression of the endogenous T-cell response to GAD.
  • the effective amount of GAD peptide or polypeptide is 0.1 ⁇ g to 10 mg per dose and preferably 1.0 ⁇ g to 1 mg per dose.
  • a dose may comprise a single administration or an administration protocol.
  • Administration may be by any convenient means such as, but not limited to, intravenous, subcutaneous, epicutaneous, infusion, oral, topical, intranasal, supository or intraperitoneal administration.
  • the GAD peptide or polypeptide may be administered alone or in combination with one or more other active molecules, molecules which facilitate the GAD peptide or polypeptide activity such as cytokines, and in particular, TNF- ⁇ and/or TNF- ⁇ .
  • the present invention contemplates the use of a peptide or polypeptide corresponding to the 67K isoform of GAD, or antigenic fragments or derivatives thereof, to measure reactivity of a patient's cells to the IDDM autoantigen.
  • the peptide or polypeptide, or fragments or derivatives thereof may be added, in solution or bound to a solid support together with cells from a patient derived from peripheral blood or from tissue biopsies either unfractionated, fractionated or derived as a continuous cell line.
  • Reactivity to the autoantigen may then be measured by standard proliferation assays such as incorporation of tritiated thymidine, standard cytotoxic assays such as release of marker radioactivity from target cells, measurements of expressed or secreted molecules such as cytokines or other standard assays of cellular reactivity which are well known in the art.
  • standard proliferation assays such as incorporation of tritiated thymidine
  • standard cytotoxic assays such as release of marker radioactivity from target cells
  • measurements of expressed or secreted molecules such as cytokines or other standard assays of cellular reactivity which are well known in the art.
  • a diagnostic kit for assaying patient T-cells Standard 96 well plates, as used in ELISA assays, are pre-coated with a monoclonal antibody (MAb) to a T-cell cytcokine such as ⁇ -interferon ( ⁇ -IFN) with or without antigen. Alternatively, antigen is added in soluble form together with aliquots of peripheral blood mononuclear cells or T-cells.
  • MAb monoclonal antibody
  • ⁇ -IFN ⁇ -interferon
  • antigen is added in soluble form together with aliquots of peripheral blood mononuclear cells or T-cells.
  • Incubation is allowed to proceed for two or more days, the cells are washed off, wells washed again and plates developed with a labelled second MAb to the cytokine such as anti- ⁇ -FN conjugated with alkaline phosphatase or horseradish peroxidase. Colorimetric reaction and read-out systems can then be utilised. Alternatively, it is possible to visualise microscopically individual spots on bottoms of wells representing cytokine produced at the single T-cell level, thereby enabling the precursor frequency of antigen-reactive T-cells to be determined.
  • a labelled second MAb to the cytokine such as anti- ⁇ -FN conjugated with alkaline phosphatase or horseradish peroxidase.
  • Colorimetric reaction and read-out systems can then be utilised.
  • kits and diagnostic assays including a kit comprising a container adapted to contain a synthetic peptide or polypeptide corresponding to the 67 isoform of GAD or its fragments, derivatives, homologues and/or immunological relatives.
  • the kit may contain a second container adapted to contain or receive a sample to be tested.
  • a third container may be present adapted to contain reagents for detecting GAD-antibody complexes.
  • the kit may comprise one or more containers (e.g. wells) adapted to contain a GAD specific antibody (e.g. a monoclonal antibody). Additional containers with the kit may then contain recepticles for receiving fluid samples and a labelled antibody.
  • expression of the cDNA insert encoding the GAD's described herein or fragments thereof may be achieved in a number of different ways.
  • successful expression of the autoantigen as a fusion protein can be achieved using the pGEX vectors which give expression of glutathione S-transferase fusion proteins, using E. coli as the host cells. Expression could also be achieved, by way of example, using the well-known pEV vectors or the, polyhistidine expression vectors (23) again using E. coli as the host cells. Alternatively, GAD may be expressed as a non-fused polypeptide, by using appropriate vector and host cell combinations.
  • Other vector and host cell combinations which can be used in accordance with the present invention including a number of well described yeast shuttle vectors for use in yeast cells, or eukaryotic vectors useful in continuous cell lines, (eg CHO cells) or transgenic animals.
  • Mouse RNA was obtained from brains of BALB/C mice.
  • RNA was obtained from human adult brain and pancreatic islets. Islets were isolated from a donor pancreas by an intraductal collagenase distension procedure. Individually hand-picked islets were lysed in 5M guanidinium isothiocynate, 10 mM Tris pH 7.6, 10 mM EDTA and RNA purified by centrifugation through a 5.7M CsCl cushion. Total RNA from human brain was a gift of Claude Bernard from Latrobe University School of Behavioural Science, Australia.
  • Human cDNA libraries Two ⁇ gt-11 based human cDNA expression libraries were used as a source of GAD cDNA.
  • a brain-stem cDNA library was purchased from Clonetech and the islet-cell library was a gift of Alan Permutt from the Washington School of Medicine, St. Louis.
  • cDNA was prepared from phage stocks by a plate lysis method (20).
  • PCR Polymease chain reaction
  • oligonucleotide primers were designed from conserved regions. The primers used to isolate the various clones are shown in Table 1.
  • First strand synthesis of total RNA (1 ⁇ g) was performed in 10 mM Tris pH 8.3, 50 mM KCl, 1.5 mM MgCl 2 , 100 ⁇ M dNTPs (PCR buffer) containing 2 pmole of complimentary primer, 40 U of RNasin and 5 U of MoMLV reverse transcriptase at 37° C. for 30 min in a 50 ⁇ l reaction volume.
  • ⁇ gt-11 cDNA 100 ng or 10 ⁇ l of the first strand reaction was amplified in PCR buffer containing 20 pmole of each primer and 2.5 U of TaqI polymerase by 30 thermal cycles (one cycle: 1.5 min at 95° C.; 2.0 min at 37°-45° C.; 2.0 min at 72° C.). Reactions were analysed on low melting agarose gels and products of the expected size purified by phenol extraction (20).
  • PCR amplified DNA fragments were cloned into the plasmid expression vector pGEX 1-3(21) and also into the histidine expression vector pDS56,(-1) and (-2) (23).
  • Nucleotide sequence was determined by the dideoxy chain termination method (22) using the M13 universal primer and specific primers (RGAD 1, SEQ ID NO.: 15; RGAD2, SEQ ID NO.: 16; RGAD3, SEQ ID NO.: 17; RGAD4, SEQ ID NO.: 18; RGAD5, SEQ ID NO.: 19; RGAD6, SEQ ID NO.: 20; GAD1, SEQ ID NO.: 21; GAD5, SEQ ID NO.: 22; GAD7, SEQ ID NO.: 23) designed from internal GAD sequence, as described in Table 1.
  • oligonucleotide pairs overlapping nucleotide stretches conserved between rat and feline sequences were synthesised and used in PCR reactions to amplify cDNA extracted from brain and islet ⁇ t-11 expression libraries as well as from RNA extracted from human brain or human islets.
  • extensive PCR reactions using various combinations of oligonucleotide primers and temperatures of annealing a product of 600 nucleotides was obtained from both brain and islet cDNA templates with the oligonucleotides primers:
  • FIG. 2 shows the deduced amino acid sequences of HBGAD and HIGAD and their alignment with the corresponding region in the feline GAD protein (aa 218-393).
  • the four nucleotide differences between HBGAD and HIGAD would result in three conservative amino acid changes at residues 247 (leucine ⁇ isoleucine) and 260 (threonine ⁇ isoleucine) and 248 (phenylalanine ⁇ leucine); residue 339 (leucine) remains unchanged because the nucleotide difference at position 366 is silent.
  • pancreatic islets Infiltration of the pancreatic islets with mononuclear cells culminates in the destruction of insulin-producing ⁇ cells and clinical IDDM (20).
  • the enzyme GAD has recently been identified as a putative islet autoantigen in IDDM based on the ability of several IDDM sera to co-precipitate the 64K islet cell protein and GAD (14) and it has been shown that peripheral blood T cells from subjects with pre-clinical and clinical IDDM can be activated by islet membrane preparations containing the 64K autoantigen and GAD (24, 25).
  • the finding of sequence differences between brain and islet GAD may now provide a genetic basis for selective autoimmune destruction of pancreatic islets.
  • GAD 5 SEQ ID NO: 22 5' CCCATAAACTCATGTTCTTG 3'
  • GAD 7 SEQ ID NO: 23 5' GGAGAAAATATCCCATCACC 3'
  • amplification of the GAD7 and GAD5 first strand products by PCR using GAD specific oligonucleotides generated a cDNA encoding aa 1-250 HBGAD17 and an overlapping cDNA that encodes aa 208-594.
  • One hundred nanograms of each fragment was denatured at 95° C.
  • Mouse Brain GAD was cloned as described above for HBGAD and HIGAD except that primers RGAD1 and RGAD6 (Table 1) were used.
  • PBMC Peripheral blood mononuclear cells
  • the recombinant GAD fusion proteins HBGAD and HIGAD which contain the 196 amino acid middle portions of human brain and human islet GAD respectively, as described in Table 1 were added to final concentrations of 10, 1.0 and 0.1 ⁇ g/ml, together with glutathione-S-transferase (GST) to which the recombinant GAD antigen is fused.
  • GST glutathione-S-transferase
  • Sonicated fetal pig islets which the present inventors have shown to contain GAD (24), as well as fetal pig liver, thyroid and kidney were also used as sources of antigen(s).
  • the cultures were incubated for 5 days in a humidified 5% CO 2 atmosphere with the addition of 3 H-thymidine (1 ⁇ Ci/well) for the last 17 hours. The cells were then harvested for scintillation counting. Median counts per minute (cpm) of each quadruplicate were used to derive stimulation indices, ie. cpm with antigen/cpm without antigen. A positive result was defined as a stimulation index greater than that obtained with GST (recombinant GAD proteins or greater than 2.0 (fetal tissues).
  • Sera samples from subjects were tested for an antibody response to the N-terminal fragment of recombinant murine brain GAD, MBGAD12 as well as against the full length recombinant human brain GAD.
  • Recombinant mouse brain GAD12 was cloned and expressed as a fusion protein with glutathione-S-transferase (GST) in the pGEX system.
  • MBGAD12 was cleaved with thrombin and the GAD portion affinity purified from GST using glutathione agarose beads.
  • MBGAD34, MBGAD56, HBGAD17 and HBGAD65 were cloned and expressed as fusion proteins with six histidine residues at the N-terminus using the polyhistidine expression system.
  • the recombinant GAD proteins were coated at 1 ⁇ g/mL on plastic wells of a 96-well plate, wells were exposed to blocking buffer, washed and incubated with doubling dilutions of test sera, washed and exposed to alkaline phosphatase--conjugated second antibody, washed, developed with n-nitrophenol chromogen and read at 405 nM. An OD>mean+2 SD with control sera was taken as positive.
  • Solimena M., Folli, F., Denis-Donini, S., Comi, G. C., Pozza, G., DeCamllli, P., and Vicari, A. M. (1988) N. Engl. J. Med. 318, 1012-1020.

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Abstract

The present invention relates to the identification, cloning and sequencing of nucleic acid molecules encoding an isoform of the enzyme glutamic acid decarboxylase and further relates to the use of these molecules and/or peptides and polypeptides encoded thereby in diagnostic tests for Insulin Dependent Diabetes Mellitus and other diseases in which glutamic acid decarboxylase is an autoantigen and in the treatment of patients suffering from these diseases.

Description

This is a continuation of application Ser. No. 07/839,805 filed on Feb. 21, 1992, now abandoned.
FIELD OF INVENTION
The present invention relates to the identification, cloning and sequencing of nucleic acid molecules encoding an isoform of the enzyme glutamic acid decarboxylase and further relates to the use of these molecules and/or peptides and polypeptides encoded thereby in diagnostic tests for Insulin Dependent Diabetes Mellitus and other diseases in which glutamic acid decarboxylase is an autoantigen and in the treatment of patients suffering from these diseases.
BACKGROUND TO THE INVENTION
The enzyme glutamic acid decarboxylase (hereinafter referred to as "GAD") catalyses the conversion of L-glutamic acid to the inhibitory neurotransmitter γ-amino butyric acid (hereinafter referred to as "GABA"). GAD is expressed both in the GABA secretory neurons of the central nervous system (1-3), in the β-cells of the pancreas (4,5), and in spermatoza (6). Analysis of imtmunoaffinity-purified, enzymatically active brain GAD has identified several isomeric forms of GAD with Mr 54-67,000 (7,8). Using antisera raised to purified brain GAD to screen brain cDNA expression libraries, cDNAs encoding full length rat (9) and feline (10) GAD sequences have been isolated and sequenced. Comparisons of the deduced amino acid sequences of rat and feline GAD show that both proteins are 95% identical and, therefore, highly conserved during evolution.
Autoantibodies reactive with GAD in GABA-ergic neurons are present in the majority of sera from patients with the rare neurological disease Stiff Man Syndrome (hereinafter referred to "SMS"; 11,12). Patients positive for GAD autoantibodies have an increased frequency of polyendocrine autoimmunity especially Insulin Dependent Diabetes Mellitus (hereinafter referred to as "IDDM"). During the pre-clinical stage of IDDM and in patients with recent onset clinical IDDM, autoantibodies are frequently detected against an islet cell Mr 64,000 protein designated "64K" (13). In a recent report, the 64K autoantigen was presumptively identified as GAD (14). However, Genovese (15) has suggested that GAD is co-precipitated with a separate 64K protein, the latter distinguished by tryptic products of Mr 37,000/40,000 that are distinct from a Mr 50,000 product of GAD. GAD comprises at least two isoforms encoded by separate genes (16, 17, 18). The predicted molecular weights of the known isoforms are approximately 67,000 and 65,000 (referred to as the "67K" and "65K" isoforms, respectively). The distribution of GAD isoforms in different tissues in still not well defined, but it is likely that the 65K isoform accounts for the GAD component of the 64K autoantigen (17).
In work leading up to the present invention, the inventors sought to clone the 67K isoform of GAD from human and other species for potential diagnostic and/or therapeutic use. In accordance with the present invention, human brain (HB), human pancreatic islet (HI) and mouse brain (MB) GAD (hereinafter referred to as "HBGAD", "HIGAD" and "MBGAD", respectively) have been cloned and sequenced. In further accordance with the present invention, recombinant GAD proteins corresponding to the 67K isoform and their fragments and derivatives were used as an antigen to detect antibodies and T-cells reactive with GAD thereby forming a basis for a new range of diagnostics and therapeutics for diseases of the type including preclinical and clinical IDDM and SMS and other diseases in which GAD is an autoantigen.
SUMMARY OF THE INVENTION
Accordingly, a first aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence which encodes or is complementary to a sequence which encodes human or mouse glutamic acid decarboxylase (GAD) corresponding to a 67K isoform of the enzyme or antigenically active fragments or derivatives thereof.
Another aspect of the present invention provides a synthetic peptide or polypeptide displaying the antigenicity of all or a portion of the 67K isoform of GAD or a fragment thereof and reactive with autoantibodies and/or T-cells.
Yet another aspect of the present invention contemplates a method for the detection of antibodies to GAD in a sample which method comprises contacting a peptide or polypeptide corresponding to all or an antigenic portion of the 67K isoform of GAD with said sample for a time and under conditions sufficient for a complex to form between the peptide or polypeptide and an antibody reactive to GAD and then detecting the complex.
Still yet another aspect of the present invention provides a method for detecting diseases of the type including IDDM and SMS, or for screening asymptomatic individuals, by the detection and/or determination of the titre of autoantibodies in a biological sample from said individual, said method comprising contacting said sample with a peptide or polypeptide corresponding to all or an antigenic portion of the 67K isoform of GAD for a time and under conditions sufficient to form a complex between the peptide or polypeptide and an antibody reactive to GAD and then detecting the complex and/or the amount of peptide or polypeptide which has been bound in a complex
Even yet another method of reducing autoantibodies and/or autoreactive T-cells to GAD in a patient in need thereof and/or to desensitise or induce tolerance to eliminate or diminish reactivity of autoreactive T-cells or autoantibodies to the autoantigen, said method comprising administering to said patient an effective amount of an antigenic peptide or polypeptide corresponding to all or part of the 67K isoform of GAD.
The present invention also provides a method of reducing autoantibodies and/or autoreactive T-celis to GAD in a patient in need thereof and/or to desensitise or induce tolerance to eliminate or diminish reactivity of autoreactive T-cells or autoantibodies to the autoantigen, said method comprising administering to said patient GAD reactive T-cell lines or clones or cell membranes and/or receptors for the antigen from said GAD reactive T-cell lines or clones for a time and under conditions sufficient to act as immunogens to induce inhibition and/or reduction of T-cell responses to GAD autoantigen.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows a comparison of the DNA sequences corresponding to human brain GAD (HBGAD); SEQ ID NO.:1 and human islet GAD (HIGAD); SEQ ID NO.:2.
FIG. 2 shows the deduced amino acid sequences of HBGAD (SEQ ID NO.:3) and HIGAD (SEQ ID NO.:4) and their alignment with the equivalent region in the feline GAD (SEQ ID NO.:5) (amino acids 218-398).
FIG. 3 shows the nucleotide sequence and deduced amino acid sequence corresponding to the full length mouse brain GAD (MBGAD) (SEQ ID NO.:6).
FIG. 4 shows the nucleotide sequence and deduced amino acid sequence corresponding to the N-terminal fragment of MBGAD designated MBGAD12 (SEQ ID NO.:7) that encodes amino acids 1-204 of the published feline GAD sequence (10).
FIG. 5 shows the nucleotide sequence and deduced amino acid sequence corresponding to the mid-region fragment of MBGAD, designated MBGAD34 (SEQ ID NO.:8) corresponding to amino acids 198-404 of the published feline GAD sequence.
FIG. 6 shows the nucleotide sequence and deduced amino acid sequence corresponding to the C-terminal fragment of MBGAD, designated MBGAD56 (SEQ ID NO.:9) corresponding to amino acids 392-593 of the published feline GAD sequence.
FIG. 7 shows the full length nucleotide sequence and deduced amino acid sequence corresponding to human brain GAD (HBGAD-FL); (SEQ ID NO.:10).
FIG. 8 shows the nucleotide sequence and deduced amino acid sequence corresponding to the N-terminal fragment of HBGAD, designated HBGAD17 (SEQ ID NO.:11) corresponding to amino acids 1-250 of the published feline GAD sequence.
FIG. 9 shows the nucleotide sequence and deduced amino acid sequence corresponding to the mid region fragment of HBGAD or HIGAD, designated HBGAD14 or HIGAD14 (SEQ ID NO.:12) corresponding to amino acids 208-404 of the published feline GAD sequence.
FIG. 10 shows the nucleotide sequence and deduced amino acid sequence corresponding to the C-terminal region fragment of HBGAD, designated HBGAD65 (SEQ ID NO.:13), corresponding to amino acid 392-594 of the published feline GAD sequence.
FIG. 11 shows the full length nucleotide sequence and deduced amino acid sequence corresponding to human islet GAD (HIGAD-FL); (SEQ ID NO.:14).
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a nucleic acid molecule comprising a nucleotide sequence which encodes or is complementary to a sequence which encodes human or mouse glutamic acid decarboxylase (GAD) corresponding to a 67K isoform of the enzyme or antigenically active fragments or derivatives thereof.
By the "67K isoform" is meant the form of GAD having approximately Mr 67,000 and/or any fragments, derivatives, homologues and/or immunological relatives thereof and which are distinguishable and/or otherwise distinct from the Mr 65,000 form of GAD and which reacts preferentially to T-cells and/or autoantibodies from individuals with clinical or preclinical IDDM, SMS and/or other similar diseases.
Preferably, the GAD is human pancreatic islet GAD (HIGAD), human brain cell GAD (HBGAD) and/or mouse brain cell GAD (MBGAD). Preferably, the nucleic acid molecule is DNA, at least a part of which has a nucleotide sequence substantially corresponding to the sequence shown in FIGS. 1, 3, 7 and/or 11 or a fragment, derivative, homologue or or immunological relative thereof or one or more sequences complementary thereto. The present invention, however also extends to any single or multiple nucleotide substitutions, deletions and/or additions to the sequence shown in FIGS. 1, 3, 7 and/or 11 and which still encode a GAD or fragment or derivative thereof having the requisite antigenic profile and reactive with autoantibodies or T-cells. Furthermore, when the nucleic acid molecule is RNA, the ribonucleotide sequence will, in a preferred embodiment, be substantially complementary to one or more of the sequences shown in FIGS. 1, 3, 7 and/or 11 or a fragment, derivative, or homolgue thereof.
The present invention also provides a recombinant nucleic acid (e.g. DNA) molecule comprising a nucleotide sequence as described above operably linked to an expression control sequence. Such a recombinant molecule may, for example, comprise an expression vector. The present invention further extends to a host cell such as a bacterium, yeast, mammalian or insect cell transformed with such a recombinant molecule. A preferred mammalian cell line is the Chinese Hamster Ovary (CHO) cell line.
Another aspect of this invention is directed to a synthetic (e.g. recombinant) peptide or polypeptide displaying the antigenicity of all or a portion of an isoform of GAD which is reactive with autoantibodies and/or T-cells.
Such a synthetic peptide or polypeptide may, for example, be prepared by recombinant means such as by the expression of a host cell transformed with the recombinant molecules described above. The peptide or polypeptide may be fused to another peptide or polypeptide. Alternatively, it may be prepared by chemical synthesis, such as by the well-known Merrifield solid-phase synthesis procedure. The synthetic (eg. recombinant) peptide or polypeptide may or may not retain GAD enzymatic activity. Furthermore, although synthetic GAD or fragments thereof represent a preferred embodiment, the present invention also emends to biologically pure preparations of the naturally occurring enzyme or its fragrments. By "biologically pure" is meant a preparation of at least 60%, preferably at least 70%, more preferably at least 80% and still more preferably at least 90% by weight enzyme.
In a most preferred embodiment, the present invention extends to naturally occurring or synthetic peptide or polypeptides corresponding to MBGAD, HIGAD and/or HBGAD and to nucleotide sequences coding for same as well as to fragments, derivatives, homolgoues or immunological relatives thereof. By way of example, such fragments are shown in FIGS. 2, 4, 5, 6, 8, 9 and 10. By "derivatives" is meant to include any single or multiple amino acid substitution, deletion and/or addition relative to the naturally occurring sequence or to the sequence as shown in FIGS. 1, 3, 7 and/or 11 and including any single or multiple substitution, deletion and/or addition to other molecules associated with the peptide or polypeptide including carbohydrate lipid and/or other proteinacious moieties. Such derivatives, therefore, include glycosylated or non-glycosylated forms or molecules with altered glyclosylation patterns.
The present invention also contemplates a method for the detection of autoantibodies associated with IDDM which method comprises contacting a peptide or polypeptide corresponding to all, or an antigenic portion of, GAD, which GAD corresponds to the 67K isoform of the enzyme, or a fragment or derivative thereof with a biological sample from a patient to be tested for a time and under conditions sufficient for a complex to form between the peptide or polypeptide and an antibody reactive to GAD and then detecting the complex. Preferably, the biological sample is serum. Even more preferably, the peptide or polypeptide is immobilised onto a solid support before, during or after contact with the serum. Methods of detection are well known and include colorimetric, fluorometric and radioactive procedures.
Other detection means can also be used such as involving agglutination. This assay can be varied in any number of ways without departing from the scope of the present invention.
The present invention also extends to the use of a peptide or polypeptide corresponding to the 67K isoform of GAD, or antigenic fragments thereof, as an antigen in a diagnostic test for diseases of the type including IDDM and SMS, or for screening asymptomatic individuals by detection or determination of the titre of antibodies in a patient's serum, for example using ELISA or RIA technology or an agglutination assay using antigen-coated beads or the like.
This aspect of the present invention may conveniently be carried out by the detection and/or determination of the titre of autoantibodies in a biological sample (e.g. serum) from a human subject, said method comprising contacting said sample with a peptide or polypeptide corresponding to an antigenic portion of the 67K isoform of GAD or a fragment or derivative thereof for a time and under conditions sufficient for a complex to form between the peptide or polypeptide and an antibody reactive to GAD and then detecting the complex and/or amount of peptide or polypeptide which has been bound in the complex. Preferably, the peptide or polypeptide is immobilised onto a solid support before, during or after contact with the sample and the peptide or polypeptide is as hereinbefore defined.
Alternatively, such diseases may be detected or at least a negative result re-confirmed or otherwise by screening for GAD associated immune complexes. It is possible, for example, that a negative autoantibody result could have been caused by autoantibodies forming complexes with GAD thereby not being available for binding in the aforementioned assay. To conveniently detect GAD immune complexes, serun or other biological fluid is contacted with an anti-GAD antibody (e.g. a monoclonal antibody) for a time and under conditions sufficient for a GAD-autoantibody immune complex to bind.
Preferably, the anti-GAD antibody is first immobilised onto a solid support. An anti-immunoglobulin antibody, generally with a label or other reporter molecule attached, is then used to screen for the antibody component of the GAD complex.
One skilled in the art will immediately recognise that the assays as contemplated herein may be modified without departing from the scope of the present invention. All such modifications and variations of these assays are encompassed by the present invention.
The invention also extends to use of the peptides and/or polypeptides, or fragments, or derivatives of the present invention in the treatment of patients. In this later aspect, such methods of treatment include their use as an adsorbent to remove autoantibodies or autoreactive cells from a patient, their use in direct administration to a patient as a means of desensitising or inducing tolerance to eliminate or diminish reactivity of autoreactive T-cells or autoantibodies to the IDDM autoantigen or to generate T-cell lines or clones to be used for or as therapeutic agents.
As contemplated herein, the method of treatment includes but is not limited to the following examples of treatment. A first example of treatment is desensitisation or tolerance induction using an effective amount of GAD peptide or polypeptide or fragments thereof to alter T-cell recognition of GAD and induce T-cell suppression. This may be achieved by using the known effect of certain ultraviolet wavelengths, especially UV-B, to modify antigen presentation through the skin (see 19). Effective amounts of GAD peptide or polypeptide or fragments thereof would be applied epicutaneously to the skin of subjects exhibiting peripheral blood T-cell reactivity to GAD, after exposure of skin to UV-B radiation. Treatment would be repeated until such time that T-cell reactivity to GAD was suppressed. A second treatment involves application of GAD to the skin together with one or more cytokines such as but not limited to TNFα or β. A third treatment involves T-cell immunisation whereby T-cell lines are generated to GAD peptide or polypeptide or fragments thereof by standard procedures, calls attenuated by fixation with agents such as glutaraldehyde or paraformaldehyde, washed under sterile conditions and re-injected to patients for a time and under conditions causing suppression of the endogenous T-cell response to GAD. These approaches of treatment are applicable to the prevention of clinical IDDM in asymptomatic subjects with preclinical IDDM or subjects with recent onset clinical IDDM, as well as to the recurrence of IDDM in subjects who have received pancreas islet cell or insulin-producing cell transplants. These approaches are also applicable to SMS and other diseases where GAD is an autoantigen. In accordance with the present invention the effective amount of GAD peptide or polypeptide is 0.1 μg to 10 mg per dose and preferably 1.0 μg to 1 mg per dose. A dose may comprise a single administration or an administration protocol. Administration may be by any convenient means such as, but not limited to, intravenous, subcutaneous, epicutaneous, infusion, oral, topical, intranasal, supository or intraperitoneal administration. The GAD peptide or polypeptide may be administered alone or in combination with one or more other active molecules, molecules which facilitate the GAD peptide or polypeptide activity such as cytokines, and in particular, TNF-α and/or TNF-β.
In yet a further embodiment, the present invention contemplates the use of a peptide or polypeptide corresponding to the 67K isoform of GAD, or antigenic fragments or derivatives thereof, to measure reactivity of a patient's cells to the IDDM autoantigen. The peptide or polypeptide, or fragments or derivatives thereof, may be added, in solution or bound to a solid support together with cells from a patient derived from peripheral blood or from tissue biopsies either unfractionated, fractionated or derived as a continuous cell line. Reactivity to the autoantigen may then be measured by standard proliferation assays such as incorporation of tritiated thymidine, standard cytotoxic assays such as release of marker radioactivity from target cells, measurements of expressed or secreted molecules such as cytokines or other standard assays of cellular reactivity which are well known in the art.
In one embodiment of this aspect of this invention there is provided a diagnostic kit for assaying patient T-cells. Standard 96 well plates, as used in ELISA assays, are pre-coated with a monoclonal antibody (MAb) to a T-cell cytcokine such as γ-interferon (γ-IFN) with or without antigen. Alternatively, antigen is added in soluble form together with aliquots of peripheral blood mononuclear cells or T-cells. Incubation is allowed to proceed for two or more days, the cells are washed off, wells washed again and plates developed with a labelled second MAb to the cytokine such as anti-γ-FN conjugated with alkaline phosphatase or horseradish peroxidase. Colorimetric reaction and read-out systems can then be utilised. Alternatively, it is possible to visualise microscopically individual spots on bottoms of wells representing cytokine produced at the single T-cell level, thereby enabling the precursor frequency of antigen-reactive T-cells to be determined.
The present invention encompasses other forms of kits and diagnostic assays including a kit comprising a container adapted to contain a synthetic peptide or polypeptide corresponding to the 67 isoform of GAD or its fragments, derivatives, homologues and/or immunological relatives. The kit may contain a second container adapted to contain or receive a sample to be tested. A third container may be present adapted to contain reagents for detecting GAD-antibody complexes. Alternatively, where the kit is to detect GAD immune complexes, the kit may comprise one or more containers (e.g. wells) adapted to contain a GAD specific antibody (e.g. a monoclonal antibody). Additional containers with the kit may then contain recepticles for receiving fluid samples and a labelled antibody.
In further accordance with the present invention, expression of the cDNA insert encoding the GAD's described herein or fragments thereof, may be achieved in a number of different ways.
As an example, successful expression of the autoantigen as a fusion protein can be achieved using the pGEX vectors which give expression of glutathione S-transferase fusion proteins, using E. coli as the host cells. Expression could also be achieved, by way of example, using the well-known pEV vectors or the, polyhistidine expression vectors (23) again using E. coli as the host cells. Alternatively, GAD may be expressed as a non-fused polypeptide, by using appropriate vector and host cell combinations. Other vector and host cell combinations which can be used in accordance with the present invention including a number of well described yeast shuttle vectors for use in yeast cells, or eukaryotic vectors useful in continuous cell lines, (eg CHO cells) or transgenic animals.
The present invention will now be further described with reference to the following non-limiting Examples.
EXAMPLE 1 Materials and Methods
Mouse RNA. Mouse RNA was obtained from brains of BALB/C mice.
Humnan RNA. RNA was obtained from human adult brain and pancreatic islets. Islets were isolated from a donor pancreas by an intraductal collagenase distension procedure. Individually hand-picked islets were lysed in 5M guanidinium isothiocynate, 10 mM Tris pH 7.6, 10 mM EDTA and RNA purified by centrifugation through a 5.7M CsCl cushion. Total RNA from human brain was a gift of Claude Bernard from Latrobe University School of Behavioural Science, Australia.
Human cDNA libraries Two λgt-11 based human cDNA expression libraries were used as a source of GAD cDNA. A brain-stem cDNA library was purchased from Clonetech and the islet-cell library was a gift of Alan Permutt from the Washington School of Medicine, St. Louis. cDNA was prepared from phage stocks by a plate lysis method (20).
Polymease chain reaction (PCR). Based on the published rat (9) and feline (10) GAD cDNA sequences, oligonucleotide primers were designed from conserved regions. The primers used to isolate the various clones are shown in Table 1. First strand synthesis of total RNA (1 μg) was performed in 10 mM Tris pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 100 μM dNTPs (PCR buffer) containing 2 pmole of complimentary primer, 40 U of RNasin and 5 U of MoMLV reverse transcriptase at 37° C. for 30 min in a 50 μl reaction volume. λgt-11 cDNA (100 ng) or 10 μl of the first strand reaction was amplified in PCR buffer containing 20 pmole of each primer and 2.5 U of TaqI polymerase by 30 thermal cycles (one cycle: 1.5 min at 95° C.; 2.0 min at 37°-45° C.; 2.0 min at 72° C.). Reactions were analysed on low melting agarose gels and products of the expected size purified by phenol extraction (20).
Cloning and DNA sequencing. PCR amplified DNA fragments were cloned into the plasmid expression vector pGEX 1-3(21) and also into the histidine expression vector pDS56,(-1) and (-2) (23). Nucleotide sequence was determined by the dideoxy chain termination method (22) using the M13 universal primer and specific primers (RGAD 1, SEQ ID NO.: 15; RGAD2, SEQ ID NO.: 16; RGAD3, SEQ ID NO.: 17; RGAD4, SEQ ID NO.: 18; RGAD5, SEQ ID NO.: 19; RGAD6, SEQ ID NO.: 20; GAD1, SEQ ID NO.: 21; GAD5, SEQ ID NO.: 22; GAD7, SEQ ID NO.: 23) designed from internal GAD sequence, as described in Table 1.
                                  TABLE 1                                 
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ORIGIN OF GAD cDNA CLONES                                                 
      AA REGION                                                           
GAD   EQUIVALENT TO                                                       
               PCR                                                        
CLONE FELINE GAD                                                          
               SOURCE      OLIGONUCLEOTIDES 5'-3'                         
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MBGAD 12                                                                  
       1-204   BRAIN RNA   RGAD1 SEQ ID NO:15 ATTGGATCCACCGAGCTGATGGCGTCTT
                           C                                              
                           RGAD2 SEQ ID NO:16 CCGAATTCGCCATTCGCCAGCTAAACC 
                           1                                              
MBGAD34                                                                   
      198-404  BRAIN RNA   RGAD3 SEQ ID NO:17 ATTGGATCCGGTTTAGCTGGCGAATGGC
                           7                                              
                           RGAD4 SEQ ID NO:18 CCGAATTCTGTGAGGGTTCCAGGTGAC 
                           .                                              
MGAD56                                                                    
      392-593  BRAIN RNA   RGAD5 SEQ ID NO:19 ATTGGATCCGTCACCTGGAACCCTCACA
                           RGAD6 SEQ ID NO:20 CCGAATTCATTACAGATCCTGGCCCA  
HBGAD 208-404  BRAIN cDNA LIBRARY                                         
                           GAD1 SEQ ID NO:21 ACTGCCAATACCAATATGTTCACATATGA
                           RGAD4 SEQ ID NO:22 CCCAATTCTGAGGGTTCCAGGTGAC   
HIGAD 208-404  ISLET cDNA LIBRARY                                         
                           GAD1 SEQ ID NO:21 ACTGCCAATACCAATATGTTCACATATGA
                           RGAD4 SEQ ID NO:23 CCGAATTCTCTGAGGGTTCCAGGTGAC 
HBGAD17                                                                   
       1-250   BRAIN RNA   RGAD1 SEQ ID NO:15 ATTGGATCCACCGAGCTGATGGCGTCTT
                           C                                              
                           GAD7 SEQ ID NO:23 GGAGAAAATATCCCATCACC         
HBGAD14                                                                   
      208-404  BRAIN RNA   GAD1 SEQ ID NO:21 ACTGCCAATACCAATATGTTCACATATGA
                           RGAD4 SEQ ID NO:18 CCGAATTCTGTGAGGGTTCCAGGTGAC 
HBGAD65                                                                   
      392-594  BRAIN RNA   GAD6 SEQ ID NO:25 ATTGGATCCGGCATAGAAAGGGCCAA   
                           GAD5 SEQ ID NO:22 CCCATAAACTCATGTTCTTG         
HBGAD-FL                                                                  
       1-594   BRAIN RNA   RGAD1 SEQ ID NO:15 ATTGGATCCACCGAGCTGATGGCGTCTT
                           C                                              
HIBAD-FL                                                                  
       1-594   PANCREAS RNA                                               
                           GAD5 SEQ ID NO:22 CCCATAAACTCATGTTCTTG         
HIGAD14                                                                   
      208-404  ISLET RNA   GAD1 SEQ ID NO:21 ACTGCCAATACCAATATGTTCACATATGA
                           RGAD4 SEQ ID NO:18 CCGAATTCTGTGAGGGTTCCAGGTGAC 
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EXAMPLE 2 Cloning of Human GAD
To clone human GAD cDNA, oligonucleotide pairs overlapping nucleotide stretches conserved between rat and feline sequences were synthesised and used in PCR reactions to amplify cDNA extracted from brain and islet λt-11 expression libraries as well as from RNA extracted from human brain or human islets. In extensive PCR reactions using various combinations of oligonucleotide primers and temperatures of annealing, a product of 600 nucleotides was obtained from both brain and islet cDNA templates with the oligonucleotides primers:
5' ACTGCCAATACCAATATGTTCACATATGA 3' SEQ ID NO: 21 and
5' CCGAATTCTGTAGAGGGTTCCAGGTGAC 3' SEQ ID NO.:24) (complementary, contains an Eco RI site) which would correspond to nucleotide positions 739-768 and 1312-1330 of the published feline cDNA (10), respectively, representing the middle portion of the GAD open reading frame. The two 600 nucleotide PCR products were digested with EcoRI and SmaI ligated with pGEX-3X DNA cleaved with EcoRI and SmaI and transformed into E. coli. Restriction analysis of plasmid DNA from transformants identified a human brian GAD clone (HBGAD) and an islet GAD clone (HIGAD).
The 540 nucleotide DNA sequences determined for both HBGAD and HIGAD, excluding the oligonucleotide sequences, are shown in FIG. 1. These two sequences display 90% similarity with the feline GAD sequence and therefore, confirm the identity of the human clones. Alignment of the HBGAD sequence with the HIGAD sequence showed that they were identical except for four nucleotide changes at position 88 (T-A), 91(T-C) 128(C-T) and 366(C-T).
FIG. 2 shows the deduced amino acid sequences of HBGAD and HIGAD and their alignment with the corresponding region in the feline GAD protein (aa 218-393). The four nucleotide differences between HBGAD and HIGAD would result in three conservative amino acid changes at residues 247 (leucine→isoleucine) and 260 (threonine→isoleucine) and 248 (phenylalanine→leucine); residue 339 (leucine) remains unchanged because the nucleotide difference at position 366 is silent. These amino acid differences between the middle one-third of the brain and islet GAD proteins provide evidence for the existence of isomeric forms of GAD in human tissue.
Infiltration of the pancreatic islets with mononuclear cells culminates in the destruction of insulin-producing β cells and clinical IDDM (20). The enzyme GAD has recently been identified as a putative islet autoantigen in IDDM based on the ability of several IDDM sera to co-precipitate the 64K islet cell protein and GAD (14) and it has been shown that peripheral blood T cells from subjects with pre-clinical and clinical IDDM can be activated by islet membrane preparations containing the 64K autoantigen and GAD (24, 25). The finding of sequence differences between brain and islet GAD may now provide a genetic basis for selective autoimmune destruction of pancreatic islets.
EXAMPLE 3 Construction of a Full Length Human Brain and Islet GAD cDNA
Normal brain RNA was reverse-transcribed with either GAD 5 SEQ ID NO: 22 (5' CCCATAAACTCATGTTCTTG 3') or GAD 7 SEQ ID NO: 23 (5' GGAGAAAATATCCCATCACC 3') oligonucleotides. As shown in Table 1, amplification of the GAD7 and GAD5 first strand products by PCR using GAD specific oligonucleotides generated a cDNA encoding aa 1-250 HBGAD17 and an overlapping cDNA that encodes aa 208-594. One hundred nanograms of each fragment was denatured at 95° C. in PCR buffer and hybrid molecules extended and amplified using RGAD 1 and GAD 5 oligonucleotides that anneal at the end of the hybridised molecules (Table 1) to generate a full length human GAD clone that encodes the 594aa GAD open reading frame to generate a full length HBGAD and HIGAD (FIGS. 7 and 11).
EXAMPLE 4 Cloning of Mouse Brain GAD
Mouse Brain GAD was cloned as described above for HBGAD and HIGAD except that primers RGAD1 and RGAD6 (Table 1) were used.
EXAMPLE 5 T-Cell Responses to Recombinant Proteins
67 subjects were tested for their T-cell response to HBGAD and HIGAD.
Subject backgrounds were as follows:
15 Recent onset clinical Diabetics (less than 3 months after onset of symptoms)
44 Pre-clinical Diabetics (asymptomatic first degree relatives of a person with IDDM who are positive for islet cell antibodies that react with islets in frozen sections of human pancreas)
8 Controls (normal healthy young adults)
Peripheral blood mononuclear cells (PBMC) were separated by Ficoll Hypaque density gradient centrifugation, and washed twice. The cells were then resuspended (2×106 /ml) in complete culture medium (RPMI 1640 with Hepes buffer 20 mM, penicillin 100 units/ml, streptomycin 100 μl/ml, 10-5 M 2-mercaptoethanol and 5% autologous serum) and seeded (200 μl/well) into 96 well round-bottomed microtitre plates. The recombinant GAD fusion proteins HBGAD and HIGAD which contain the 196 amino acid middle portions of human brain and human islet GAD respectively, as described in Table 1 were added to final concentrations of 10, 1.0 and 0.1 μg/ml, together with glutathione-S-transferase (GST) to which the recombinant GAD antigen is fused. Sonicated fetal pig islets, which the present inventors have shown to contain GAD (24), as well as fetal pig liver, thyroid and kidney were also used as sources of antigen(s).
The cultures were incubated for 5 days in a humidified 5% CO2 atmosphere with the addition of 3 H-thymidine (1 μCi/well) for the last 17 hours. The cells were then harvested for scintillation counting. Median counts per minute (cpm) of each quadruplicate were used to derive stimulation indices, ie. cpm with antigen/cpm without antigen. A positive result was defined as a stimulation index greater than that obtained with GST (recombinant GAD proteins or greater than 2.0 (fetal tissues).
              TABLE 2                                                     
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Reactivity of Peripheral Blood T-cells                                    
           Antigens                                                       
             H islet     H brain Fetal pig                                
Subject group                                                             
             GAD14       GAD14   proislets                                
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Recent onset 10/15        8/14    5/12                                    
clinical diabetes                                                         
Preclinical diabetes                                                      
             25/44       18/36   16/34                                    
Controls     3/8         3/8     1/8                                      
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The results shown in Table 2 indicate that overall, 35/59 (59%) recent onset or preclinical subjects have circulating T-cells capable of proliferating in response to human islet GAD and (26/50) 52% to human brain GAD.
EXAMPLE 6 Antibody Responses to Recomnbinant Proteins
Sera samples from subjects were tested for an antibody response to the N-terminal fragment of recombinant murine brain GAD, MBGAD12 as well as against the full length recombinant human brain GAD.
Protein Used As Antigen
Recombinant mouse brain GAD12 was cloned and expressed as a fusion protein with glutathione-S-transferase (GST) in the pGEX system. MBGAD12 was cleaved with thrombin and the GAD portion affinity purified from GST using glutathione agarose beads. MBGAD34, MBGAD56, HBGAD17 and HBGAD65 were cloned and expressed as fusion proteins with six histidine residues at the N-terminus using the polyhistidine expression system.
ELISAS
In all ELISA assays, the recombinant GAD proteins, were coated at 1 μg/mL on plastic wells of a 96-well plate, wells were exposed to blocking buffer, washed and incubated with doubling dilutions of test sera, washed and exposed to alkaline phosphatase--conjugated second antibody, washed, developed with n-nitrophenol chromogen and read at 405 nM. An OD>mean+2 SD with control sera was taken as positive.
Subject Patients were as follows:
The results of ELISA using MBGAD12, MBGAD34 and MBGAD56, and HBGAD17 and HBGAD65 are shown in Tables 3 and 4, respectively:
              TABLE 3                                                     
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             MBGAD12 MBGAD34   MBGAD56                                    
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Preclinical IDDM                                                          
               5/9       5/9       4/9                                    
Recent onset Clinical IDDM                                                
               2/13      4/13      3/13                                   
Controls       0/22      0/20      0/20                                   
______________________________________
Seven of nine (78%) preclinical IDDM and six of 13 (46%) recent-onset IDDM sera reacted with at least one of the MBGAD peptides. Only three of nine (33%) and one of 13 (8%) preclinical and recent onset IDDM sera, respectively, reacted with all three MBGAD fragments. None of the three GAD peptides was recognised preferentially by either sera group. These findings indicate that patterns of sera reactivity with recombinant MBGAD are heterogenous and that at least three major epitopes exist in the GAD67 isoform.
              TABLE 4                                                     
______________________________________                                    
SUBJECTS        HBGAD 17  HBGAD65                                         
______________________________________                                    
Pre-clinical IDDM                                                         
                7/9       3/9                                             
Recent onset IDDM                                                         
                3/7       3/7                                             
Controls         0/16      0/16                                           
______________________________________
The results using the two human brain GAD fragments HBGAD17 and HBGAD65 in an ELISA format are comparable with those obtained using are equivalent mouse brain GAD peptides MBGAD12 and MBGAD56.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
REFERENCES
1. Roberts, E., Chase, T. N., and Tower, D. B. (1976) Kroc Foundation Series, Vol 5; GABA in Nervous System Function, Raven Press, New York.
2. Mugaini, E., and Oertel, W. H. (1985) In Handbook of Chemical Neuroanatomy (A. Bjorklund and T. Hokfelt, Eds.) Vol 4, pp 436-608 Elsevier, New York.
3. Blessing, W. W. (1990) Neuroscience 37, 171-185.
4. Okada, Y., Taniguchi. H., and Shimada, C. (1976) Science 194, 620-622.
5. Garry, D. J., Appel, N. M., Ganry, M. G., and Sorensen, R. L. (1988) J. Histochem. Cytochem. 36, 573-580.
6. Persson, H., Pelto-Huikko, M., Metsis, M., Soder, O., Brene, S., Skog, S., Hokfelt, T., and Ritzen, E. M. (1990) Mol. Cell. Biol. 19, 4701-4711.
7. Gottlieb, D. I., Chang, Y- C., and Schwob, J. E. 9186) Proc. Natl. Acad. Sci. USA. 83, 8808-8812.
8. Chang, Y- C., and Gottlieb, D. L. (1988) J. Neuroscience 8, 2123-2130.
9. Julien, J- F., Samana, P., and Mallet, J. (1990) J. Neurochemistry 54, 703-705.
10. Kobayashi, Y., Kaufman, D. L., and Tobin, A. J. (1987) J. Neuroscience 7, 2768-2772.
11. Solimena, M., Folli, F., Denis-Donini, S., Comi, G. C., Pozza, G., DeCamllli, P., and Vicari, A. M. (1988) N. Engl. J. Med. 318, 1012-1020.
12. Solimena, M., Folli, F., Aparisi, R., Pozza, G., and DeCamilli, P. (1990) N. Engl. J. Med. 322, 1555-1560.
13. Baekkeskov, S., Nielson, J. H. Marner, B., Bilde, T., Ludvigsson, J., and Lernmark, A. (1982) Nature 298, 167-169.
14. Baekkeskov, S., Aanstoot, H- J., Christgau, S., Reetz, A, Solimena, S., Cascalho, M., Folli, F., Richter-Olesen, H., and DeCamilli, P. (1990) Nature 347, 151-156.
15. Genovese, S., Cassidy, D., Bonifacio, E., Bottazzo, G. F. and Christie, M. R. (1991) Diab. Clin. Res. Prac. 14(Suppl 1), S11.
16. Cram, D. S., Barnett, L. D., Joseph, J. L. and Harrison, L. C. (1991) Biochem. Biophys. Res. Commun. 176, 1239-1244.
17. Karlsen, A. E., Hagopian, W. Z., Crubin, C. E. et el (1991) Proc. Nad. Acad. Sci. USA 88, 8337-8341.
18. Erlander, M. G., Tillakaratne, N. J. K., Feldblum, S., Patel, N. and Tobin, A. J. (1991) Neuron 7, 91-100.
19. Ullrich S. E., Yee, C. K., Kripke, M. L. (1986) Immunology 58, 158-190.
20. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) In Molecular Cloning. A Laboratory Manual. Vol 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
21. Smith, D. B., and Johnson, K. S. (1988) Gene 67, 31-40.
22. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA. 74, 5463-5467.
23. Hochuli, E., Bamwarth, W., Dobeli, H., Gentz, R. and Stuber, D. (1988) Biotechnology 6 1321-1325.
24. Harison, L. C., De Aizpurua, H., Loudovaris, T., CampbelI, I. L., Cebon, J. S., Tait, B. D., Colman, P. G. (1991) Diabetes 40,1128-1133.
25. Harrison, L. C., Chu, X. S., De Aizpurua, H. J., Graham, M., Honeyman, M. C., Colmnan, P. G. (1992) J. Clin. Invest. (in press).
__________________________________________________________________________
SEQUENCE LISTING                                                          
(1) GENERAL INFORMATION:                                                  
(iii) NUMBER OF SEQUENCES: 25                                             
(2) INFORMATION FOR SEQ ID NO:1:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 543 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA                                                  
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                   
ATTGCACCCGTGTTTGTTCTCATGGAACAGATTACTCTTAAGAAGATGAGAAAGATCGTT60            
GGATGGTCAAATAAAGATGGTGATGGGTTATTTTCTCCTGGGGGAGCCATATCCAATATG120           
TACAGCACCATGGCTGCTCGTTACAAGTACTTCCCAGAAGTGAAGACAAAAGGCATGGCG180           
GCTGTGCCCAAACTGGTCCTCTTCACCTCAGAACACAGTCACTATTCCATAAAGAAAGCC240           
GGGGCTGCGCTTGGCTTTGGAACCGACAATGTGATTTTGATAAAGTGCAATGAAAGGGGG300           
AAGATAATTCCGGCTGATTTAGAGGCAAAAATTCTTGATGCCAAACAAAAGGGCTATGTT360           
CCCCTCTATGTCAATGCAACCGCAGGCACGACTGTTTACGGAGCATTCGATCCAATCCAG420           
GAAATTGCGGACATATGTGAGAAATACAACCTTTGGCTGCATGTGGATGCTGCCTGGGGT480           
GGTGGACTGCTCATGTCCCGGAAGCACCGCCACAAACTCAGCGGCATAGAAAGGGCCAAT540           
TCA543                                                                    
(2) INFORMATION FOR SEQ ID NO:2:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 543 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA                                                  
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                   
ATTGCACCCGTGTTTGTTCTCATGGAACAGATTACTCTTAAGAAGATGAGAAAGATCGTT60            
GGATGGTCAAATAAAGATGGTGATGGGTTACTTTCTCCTGGGGGAGCCATATCCAATATG120           
TACAGCATCATGGCTGCTCGTTACAAGTACTTCCCAGAAGTGAAGACAAAAGGCATGGCG180           
GCTGTGCCCAAACTGGTCCTCTTCACCTCAGAACACAGTCACTATTCCATAAAGAAAGCC240           
GGGGCTGCGCTTGGCTTTGGAACCGACAATGTGATTTTGATAAAGTGCAATGAAAGGGGG300           
AAGATAATTCCGGCTGATTTAGAGGCAAAAATTCTTGATGCCAAACAAAAGGGCTATGTT360           
CCCCTTTATGTCAATGCAACCGCAGGCACGACTGTTTACGGAGCATTCGATCCAATCCAG420           
GAAATTGCGGACATATGTGAGAAATACAACCTTTGGCTGCATGTGGATGCTGCCTGGGGT480           
GGTGGACTGCTCATGTCCCGGAAGCACCGCCACAAACTCAGCGGCATAGAAAGGGCCAAT540           
TCA543                                                                    
(2) INFORMATION FOR SEQ ID NO:3:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 181 amino acids                                               
(B) TYPE: amino acid                                                      
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: protein                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                   
IleAlaProValPheValLeuMetGluGlnIleThrLeuLysLysMet                          
151015                                                                    
ArgLysIleValGlyTrpSerAsnLysAspGlyAspGlyLeuPheSer                          
202530                                                                    
ProGlyGlyAlaIleSerAsnMetTyrSerThrMetAlaAlaArgTyr                          
354045                                                                    
LysTyrPheProGluValLysThrLysGlyMetAlaAlaValProLys                          
505560                                                                    
LeuValLeuPheThrSerGluHisSerHisTyrSerIleLysLysAla                          
65707580                                                                  
GlyAlaAlaLeuGlyPheGlyThrAspAsnValIleLeuIleLysCys                          
859095                                                                    
AsnGluArgGlyLysIleIleProAlaAspLeuGluAlaLysIleLeu                          
100105110                                                                 
AspAlaLysGlnLysGlyTyrValProLeuTyrValAsnAlaThrAla                          
115120125                                                                 
GlyThrThrValTyrGlyAlaPheAspProIleGlnGluIleAlaAsp                          
130135140                                                                 
IleCysGluLysTyrAsnLeuTrpLeuHisValAspAlaAlaTrpGly                          
145150155160                                                              
GlyGlyLeuLeuMetSerArgLysHisArgHisLysLeuSerGlyIle                          
165170175                                                                 
GluArgAlaAsnSer                                                           
180                                                                       
(2) INFORMATION FOR SEQ ID NO:4:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 181 amino acids                                               
(B) TYPE: amino acid                                                      
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: protein                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                   
IleAlaProValPheValLeuMetGluGlnIleThrLeuLysLysMet                          
151015                                                                    
ArgLysIleValGlyTrpSerAsnLysAspGlyAspGlyIleLeuSer                          
202530                                                                    
ProGlyGlyAlaIleSerAsnMetTyrSerIleMetAlaAlaArgTyr                          
354045                                                                    
LysTyrPheProGluValLysThrLysGlyMetAlaAlaValProLys                          
505560                                                                    
LeuValLeuPheThrSerGluHisSerHisTyrSerIleLysLysAla                          
65707580                                                                  
GlyAlaAlaLeuGlyPheGlyThrAspAsnValIleLeuIleLysCys                          
859095                                                                    
AsnGluArgGlyLysIleIleProAlaAspLeuGluAlaLysIleLeu                          
100105110                                                                 
AspAlaLysGlnLysGlyTyrValProLeuTyrValAsnAlaThrAla                          
115120125                                                                 
GlyThrThrValTyrGlyAlaPheAspProIleGlnGluIleAlaAsp                          
130135140                                                                 
IleCysGluLysTyrAsnLeuTrpLeuHisValAspAlaAlaTrpGly                          
145150155160                                                              
GlyGlyLeuLeuMetSerArgLysHisArgHisLysLeuSerGlyIle                          
165170175                                                                 
GluArgAlaAsnSer                                                           
180                                                                       
(2) INFORMATION FOR SEQ ID NO:5:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 181 amino acids                                               
(B) TYPE: amino acid                                                      
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: protein                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                   
IleAlaProValPheValLeuMetGluGlnIleThrLeuLysLysMet                          
151015                                                                    
ArgGluIleValGlyTrpSerSerLysAspGlyAspGlyIlePheSer                          
202530                                                                    
ProGlyGlyAlaIleSerAsnMetTyrSerIleMetAlaAlaArgTyr                          
354045                                                                    
LysPhePheProGluValLysThrLysGlyMetAlaAlaValProLys                          
505560                                                                    
LeuValLeuPheThrSerGluHisSerHisTyrSerIleLysLysAla                          
65707580                                                                  
GlyAlaAlaLeuGlyPheGlyThrAspAsnValIleLeuIleLysCys                          
859095                                                                    
AsnGluArgGlyLysIleIleProAlaAspLeuGluAlaLysIleLeu                          
100105110                                                                 
GluAlaLysGlnLysGlyTyrValProLeuTyrValAsnAlaThrAla                          
115120125                                                                 
GlyThrThrValTyrGlyAlaPheAspProIleGlnGluIleAlaAsp                          
130135140                                                                 
IleCysGluLysTyrAsnLeuTrpLeuHisValAspAlaAlaTrpGly                          
145150155160                                                              
GlyGlyLeuLeuMetSerArgLysHisArgHisLysLeuSerGlyIle                          
165170175                                                                 
GluArgAlaAsnSer                                                           
180                                                                       
(2) INFORMATION FOR SEQ ID NO:6:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 1782 base pairs                                               
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA to mRNA                                          
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                   
ATGGCGTCTTCCACTCCTTCGCCTGCAACCTCCTCGAACGCGGGAGCGGATCCTAATACT60            
ACCAACCTGCGCCCTACAACGTATGATACTTGGTGTGGCGTAGCCCATGGATGCACCAGA120           
AAACTGGGCCTGAAGATCTGTGGCTTCTTACAAAGGACCAATAGCCTGGAAGAGAAGAGT180           
CGTCTTGTGAGCGCCTTCAGGGAGAGGCAGTCCTCCAAGAACCTGCTTTCCTGTGAAAAC240           
AGTGACCAGGGTGCCCGCTTCCGGCGCACAGAGACCGACTTCTCCAACCTGTTTGCTCAA300           
GATCTGCTTCCAGCTAAGAACGGGGAGGAGCAAACTGCGCAGTTCTTGCTGGAAGTGGTA360           
GACATACTCCTCAACTATGTCCGCAAGACATTTGATCGCTCCACCAAGGTTCTGGATTTC420           
CACCACCCACACCAGTTGCTGGAAGGCATGGAAGGCTTTAATTTGGAGCTGTCTGACCAC480           
CCCGAGTCTCTGGAGCAGATCCTGGTTGACTGTAGAGACACCCTGAAGTACGGGGTTCGC540           
ACAGGTCACCCTCGATTTTTCAACCAGCTCTCTACTGGTTTGGATATCATTGGTTTAGCT600           
GGCGAATGGCTGACATCGACTGCCAATACCAATATGTTCACATATGAAATTGCACCCGTG660           
TTTGTTCTCATGGAACAGATTACTCTTAAGAAGATGAGAAAGATCGTTGGATGGTCAAAT720           
AAAGATGGTGATGGGATATTTTCTCCTGGGGGAGCCATATCCAATATGTACAGCATCATG780           
GCTGCTCGTTACAAGTACTTCCCAGAAGTGAAGACAAAAGGCATGGCGGCTGTGCCCAAA840           
CTGGTCCTCTTCACCTCAGAACACAGTCACTATTCCATAAAGAAAGCCGGGGCTGCGCTT900           
GGCTTTGGAACCGACAATGTGATTTTGATAAAGTGCAATGAAAGGGGGAAGATAATTCCG960           
GCTGATTTAGAGGCAAAAATTCTTGATGCCAAACAAAAGGGCTATGTTCCCCTTTATGTC1020          
AATGCAACCGCAGGCACGACTGTTTACGGAGCATTCGATCCAATCCAGGAAATTGCGGAC1080          
ATATGTGAGAAATACAACCTTTGGCTGCATGTGGATGCTGCCTGGGGTGGTGGACTGCTC1140          
ATGTCCCGGAAGCACCGCCACAAACTCAGCGGCATAGAAAGGGCCAATTCAGTCACCTGG1200          
AACCCTCACAAGATGATGGGCGTGCTGCTCCAGTGCTCTGCCATTCTGGTCAAGGAAAAG1260          
GGTATACTCCAAGGATGCAACCAGATGTGTGCAGGCTACCTCTTCCAGCCAGACAAGCAG1320          
TATGACGTCTCCTATGACACCGGGGACAAGGCGATTCAGTGTGGCCGCCATGTGGACATC1380          
TTCAAGTTCTGGCTGATGTGGAAAGCAAAGGGCACCGTGGGATTTGAAAACCAGATCAAC1440          
AAATGCCTGGAGCTGGCTGATTACCTCTACGCCAAGATTAAAAACAGAGAAGAGTTTGAG1500          
ATGGTTTTCGATGGTGAGCCTGAGCACACAAATGTCTGTTTCTGGTACATTCCACAAAGC1560          
CTTCGAGGGGTTCCAGATAGCCCTGAGCGACGAGAAAAGCTACACAGGGTGGCTCCCAAG1620          
ATCAAAGCTCTGATGATGGAGTCAGGAACAACCATGGTGGGCTACCAGCCTCAAGGGGAC1680          
AAGGCCAACTTCTTCCGGATGGTCATCTCTAACCCAGCCGCCACCCAGTCTGACATCGAT1740          
TTCCTCATTGAGGAGATAGAGAGGTTGGGCCAGGATCTGTAA1782                            
(2) INFORMATION FOR SEQ ID NO:7:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 609 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA to mRNA                                          
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                   
ATGGCGTCTTCCACTCCTTCGCCTGCAACCTCCTCGAACGCGGGAGCGGATCCTAATACT60            
ACCAACCTGCGCCCTACAACGTATGATACTTGGTGTGGCGTAGCCCATGGATGCACCAGA120           
AAACTGGGCCTGAAGATCTGTGGCTTCTTACAAAGGACCAATAGCCTGGAAGAGAAGAGT180           
CGTCTTGTGAGCGCCTTCAGGGAGAGGCAGTCCTCCAAGAACCTGCTTTCCTGTGAAAAC240           
AGTGACCAGGGTGCCCGCTTCCGGCGCACAGAGACCGACTTCTCCAACCTGTTTGCTCAA300           
GATCTGCTTCCAGCTAAGAACGGGGAGGAGCAAACTGCGCAGTTCTTGCTGGAAGTGGTA360           
GACATACTCCTCAACTATGTCCGCAAGACATTTGATCGCTCCACCAAGGTTCTGGATTTC420           
CACCACCCACACCAGTTGCTGGAAGGCATGGAAGGCTTTAATTTGGAGCTGTCTGACCAC480           
CCCGAGTCTCTGGAGCAGATCCTGGTTGACTGTAGAGACACCCTGAAGTACGGGGTTCGC540           
ACAGGTCACCCTCGATTTTTCAACCAGCTCTCTACTGGTTTGGATATCATTGGTTTAGCT600           
GGCGAATGG609                                                              
(2) INFORMATION FOR SEQ ID NO:8:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 618 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA to mRNA                                          
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                   
GGTTTAGCTGGCGAATGGCTGACATCGACTGCCAATACCAATATGTTCACATATGAAATT60            
GCACCCGTGTTTGTTCTCATGGAACAGATTACTCTTAAGAAGATGAGAAAGATCGTTGGA120           
TGGTCAAATAAAGATGGTGATGGGATATTTTCTCCTGGGGGAGCCATATCCAATATGTAC180           
AGCATCATGGCTGCTCGTTACAAGTACTTCCCAGAAGTGAAGACAAAAGGCATGGCGGCT240           
GTGCCCAAACTGGTCCTCTTCACCTCAGAACACAGTCACTATTCCATAAAGAAAGCCGGG300           
GCTGCGCTTGGCTTTGGAACCGACAATGTGATTTTGATAAAGTGCAATGAAAGGGGGAAG360           
ATAATTCCGGCTGATTTAGAGGCAAAAATTCTTGATGCCAAACAAAAGGGCTATGTTCCC420           
CTTTATGTCAATGCAACCGCAGGCACGACTGTTTACGGAGCATTCGATCCAATCCAGGAA480           
ATTGCGGACATATGTGAGAAATACAACCTTTGGCTGCATGTGGATGCTGCCTGGGGTGGT540           
GGACTGCTCATGTCCCGGAAGCACCGCCACAAACTCAGCGGCATAGAAAGGGCCAATTCA600           
GTCACCTGGAACCCTCAC618                                                     
(2) INFORMATION FOR SEQ ID NO:9:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 612 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA to mRNA                                          
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                   
GGCATAGAAAGGGCCAATTCAGTCACCTGGAACCCTCACAAGATGATGGGCGTGCTGCTC60            
CAGTGCTCTGCCATTCTGGTCAAGGAAAAGGGTATACTCCAAGGATGCAACCAGATGTGT120           
GCAGGCTACCTCTTCCAGCCAGACAAGCAGTATGACGTCTCCTATGACACCGGGGACAAG180           
GCGATTCAGTGTGGCCGCCATGTGGACATCTTCAAGTTCTGGCTGATGTGGAAAGCAAAG240           
GGCACCGTGGGATTTGAAAACCAGATCAACAAATGCCTGGAGCTGGCTGATTACCTCTAC300           
GCCAAGATTAAAAACAGAGAAGAGTTTGAGATGGTTTTCGATGGTGAGCCTGAGCACACA360           
AATGTCTGTTTCTGGTACATTCCACAAAGCCTTAGAGGGGTTCCAGATAGCCCTGAGCGA420           
CGAGAAAAGCTACACAGGGTGGCTCCCAAGATCAAAGCTCTGATGATGGAGTCAGGAACA480           
ACCATGGTCGGCTACCAGCCTCAAGGGGACAAGGCCAACTTCTTCCGGATGGTCATCTCT540           
AACCCAGCCGCCACCCAGTCTGACATCGATTTCCTCATTGAGGAGATAGAGAGGTTGGGC600           
CAGGATCTGTAA612                                                           
(2) INFORMATION FOR SEQ ID NO:10:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 1785 base pairs                                               
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA to mRNA                                          
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                  
ATGGCGTCTTCGACCCCATCTTCGTCCGCAACCTCCTCGAACGCGGGAGCGGACCCCAAT60            
ACCACTAACCTGCGCCCCACAACGTACGATACCTGGTGCGGCGTGGCCCATGGATGCACC120           
AGAAAACTGGGGCTCAAGATCTGCGGCTTCTTGCAAAGGACCAACAGCCTGGAAGAGAAG180           
AGTCGCCTTGTGAGTGCCTTCAAGGAGAGGCAATCCTCCAAGAACCTGCTTTCCTGTGAA240           
AACAGCGACCGGGATGCCCGCTTCCGGCGCACAGAGACTGACTTCTCTAATCTGTTTGCT300           
AGAGATCTGCTTCCGGCTAAGAACGGTGAGGAGCAAACCGTGCAATTCCTCCTGGAAGTG360           
GTGGACATACTCCTCAACTATGTCCGCAAGACATTTGATCGCTCCACCAAGGTGCTGGAC420           
TTTCATCACCCACACCAGTTGCTGGAAGGCATGGAGGGCTTCAACTTGGAGCTCTCTGAC480           
CACCCCGAGTCCCTGGAGCAGATCCTGGTCGACTGCAGAGACACCTTGAAGTATGGGGTT540           
CGCACAGGTCATCCTCGATTTTTCAACCAGCTCTCCACTGGATTGGATATTATTGGCCTA600           
GCTGGAGAATGGCTGACATCAACGGCCAATACCAACATGTTCACATATGAAATTGCACCA660           
GTGTTTGTCCTCATGGAACAAATAACACTTAAGAAGATGAGAGAGATAGTTGGATGGTCA720           
AGTAAAGATGGTGATGGGATATTTTCTCCTGGGGGCGCCATATCCAACATGTACAGCATC780           
ATGGCTGCTCGCTACAAGTACTTCCCGGAAGTTAAGACAAAGGGCATGGCGGCTGTGCCT840           
AAACTGGTCCTCTTCACCTCAGAACAGAGTCACTATTCCATAAAGAAAGCTGGGGCTGCA900           
CTTGGCTTTGGAACTGACAATGTGATTTTGATAAAGTGCAATGAAAGGGGGAAAATAATT960           
CCAGCTGATTTTGAGGCAAAAATTCTTGAAGCCAAACAGAAGGGATATGTTCCCTTTTAT1020          
GTCAATGCAACTGCTGGCACGACTGTTTATGGAGCTTTTGATCCGATACAAGAGATTGCA1080          
GATATATGTGAGAAATATAACCTTTGGTTGCATGTCGATGCTGCCTGGGGAGGTGGGCTG1140          
CTCATGTCCAGGAAGCACCGCCATAAACTCAACGGCATAGAAAGGGCCAACTCAGTCACC1200          
TGGAACCCTCACAAGATGATGGGCGTGCTGTTGCAGTGCTCTGCCATTCTCGTCAAGGAA1260          
AAGGGTATACTCCAAGGATGCAACCAGATGTGTGCAGGATACCTCCTCCAGCCAGACAAG1320          
CAGTATGATGTCTCCTACGACACCGGGGACAAGGCAATTCAGTGTGGCCGCCACGTGGAT1380          
ATCTTCAAGTTCTGGCTGATGTGGAAAGCAAAGGGCACAGTGGGATTTGAAAACCAGATC1440          
AACAAATGCCTGGAACTGGCTGAATACCTCTATGCCAAGATTAAAAACAGAGAAGAATTT1500          
GAGATGGTTTTCAATGGCGAGCCTGAGCACACAAACGTCTGTTTTTGGTATATTCCACAA1560          
AGCCTCAGGGGTGTGCCAGACAGCCCTCAACGACGGGAAAAGCTACACAAGGTGGCTCCA1620          
AAAATCAAAGCCCTGATGATGGAGTCAGGTACGACCATGGTTGGCTACCAGCCCCAAGGG1680          
GACAAGGCCAACTTCTTCCGGATGGTCATCTCCAACCCAGCCGCTACCCAGTCTGACATT1740          
GACTTCCTCATTGAGGAGATAGAAAGACTGGGCCAGGATCTGTAA1785                         
(2) INFORMATION FOR SEQ ID NO:11:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 750 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA to mRNA                                          
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                  
ATGGCGTCTTCGACCCCATCTTCGTCCGCAACCTCCTCGAACGCGGGAGCGGACCCCAAT60            
ACCACTAACCTGCGCCCCACAACGTACGATACCTGGTGCGGCGTGGCCCATGGATGCACC120           
AGAAAACTGGGGCTCAAGATCTGCGGCTTCTTGCAAAGGACCAACAGCCTGGAAGAGAAG180           
AGTCGCCTTGTGAGTGCCTTCAAGGAGAGGCAATCCTCCAAGAACCTGCTTTCCTGTGAA240           
AACAGCGACCGGGATGCCCGCTTCCGGCGCACAGAGACTGACTTCTCTAATCTGTTTGCT300           
AGAGATCTGCTTCCGGCTAAGAACGGTGAGGAGCAAACCGTGCAATTCCTCCTGGAAGTG360           
GTGGACATACTCCTCAACTATGTCCGCAAGACATTTGATCGCTCCACCAAGGTGCTGGAC420           
TTTCATCACCCACACCAGTTGCTGGAAGGCATGGAGGGCTTCAACTTGGAGCTCTCTGAC480           
CACCCCGAGTCCCTGGAGCAGATCCTGGTCGACTGCAGAGACACCTTGAAGTATGGGGTT540           
CGCACAGGTCATCCTCGATTTTTCAACCAGCTCTCCACTGGATTGGATATTATTGGCCTA600           
GCTGGAGAATGGCTGACATCAACGGCCAATACCAACATGTTCACATATGAAATTGCACCA660           
GTGTTTGTCCTCATGGAACAAATAACACTTAAGAAGATGAGAGAGATAGTTGGATGGTCA720           
AGTAAAGATGGTGATGGGATATTTTCTCCT750                                         
(2) INFORMATION FOR SEQ ID NO:12:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 591 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA to mRNA                                          
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                  
ACGGCCAATACCAACATGTTCACATATGAAATTGCACCAGTGTTTGTCCTCATGGAACAA60            
ATAACACTTAAGAAGATGAGAGAGATAGTTGGATGGTCAAGTAAAGATGGTGATGGGATA120           
TTTTCTCCTGGGGGCGCCATATCCAACATGTACAGCATCATGGCTGCTCGCTACAAGTAC180           
TTCCCGGAAGTTAAGACAAAGGGCATGGCGGCTGTGCCTAAACTGGTCCTCTTCACCTCA240           
GAACAGAGTCACTATTCCATAAAGAAAGCTGGGGCTGCACTTGGCTTTGGAACTGACAAT300           
GTGATTTTGATAAAGTGCAATGAAAGGGGGAAAATAATTCCAGCTGATTTTGAGGCAAAA360           
ATTCTTGAAGCCAAACAGAAGGGATATGTTCCCTTTTATGTCAATGCAACTGCTGGCACG420           
ACTGTTTATGGAGCTTTTGATCCGATACAAGAGATTGCAGATATATGTGAGAAATATAAC480           
CTTTGGTTGCATGTCGATGCTGCCTGGGGAGGTGGGCTGCTCATGTCCAGGAAGCACCGC540           
CATAAACTCAACGGCATAGAAAGGGCCAACTCAGTCACCTGGAACCCTCAC591                    
(2) INFORMATION FOR SEQ ID NO:13:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 612 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA to mRNA                                          
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                  
GGCATAGAAAGGGCCAACTCAGTCACCTGGAACCCTCACAAGATGATGGGCGTGCTGTTG60            
CAGTGCTCTGCCATTCTCGTCAAGGAAAAGGGTATACTCCAAGGATGCAACCAGATGTGT120           
GCAGGATACCTCTTCCAGCCAGACAAGCAGTATGATGTCTCCTACGACACCGGGGACAAG180           
GCAATTCAGTGTGGCCGCCACGTGGATATCTTCAAGTTCTGGCTGATGTGGAAAGCAAAG240           
GGCACAGTGGGATTTGAAAACCAGATCAACAAATGCCTGGAACTGGCTGAATACCTCTAT300           
GCCAAGATTAAAAACAGAGAAGAATTTGAGATGGTTTTCAATGGCGAGCCTGAGCACACA360           
AACGTCTGTTTTTGGTATATTCCACAAAGCCTCAGGGGTGTGCCAGACAGCCCTCAACGA420           
CGGGAAAAGCTACACAAGGTGGCTCCAAAAATCAAAGCCCTGATGATGGAGTCAGGTACG480           
ACCATGGTTGGCTACCAGCCCCAAGGGGACAAGGCCAACTTCTTCCGGATGGTCATCTCC540           
AACCCAGCCGCTACCCAGTCTGACATTGACTTCCTCATTGAGGAGATAGAAAGACTGGGC600           
CAGGATCTGTAA612                                                           
(2) INFORMATION FOR SEQ ID NO:14:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 1785 base pairs                                               
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA to mRNA                                          
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                  
ATGGCGTCTTCGACCCCATCTTCGTCCGCAACCTCCTCGAACGCGGGAGCGGACCCCAAT60            
ACCACTAACCTGCGCCCCACAACGTACGATACCTGGTGCGGCGTGGCCCATGGATGCACC120           
AGAAAACTGGGGCTCAAGATCTGCGGCTTCTTGCAAAGGACCAACAGCCTGGAAGAGAAG180           
AGTCGCCTTGTGAGTGCCTTCAAGGAGAGGCAATCCTCCAAGAACCTGCTTTCCTGTGAA240           
AACAGCGACCGGGATGCCCGCTTCCGGCGCACAGAGACTGACTTCTCTAATCTGTTTGCT300           
AGAGATCTGCTTCCGGCTAAGAACGGTGAGGAGCAAACCGTGCAATTCCTCCTGGAAGTG360           
GTGGACATACTCCTCAACTATGTCCGCAAGACATTTGATCGCTCCACCAAGGTGCTGGAC420           
TTTCATCACCCACACCAGTTGCTGGAAGGCATGGAGGGCTTCAACTTGGAGCTCTCTGAC480           
CACCCCGAGTCCCTGGAGCAGATCCTGGTCGACTGCAGAGACACCTTGAAGTATGGGGTT540           
CGCACAGGTCATCCTCGATTTTTCAACCAGCTCTCCACTGGATTGGATATTATTGGCCTA600           
GCTGGAGAATGGCTGACATCAACGGCCAATACCAACATGTTCACATATGAAATTGCACCA660           
GTGTTTGTCCTCATGGAACAAATAACACTTAAGAAGATGAGAGAGATAGTTGGATGGTCA720           
AGTAAAGATGGTGATGGGATATTTTCTCCTGGGGGCGCCATATCCAACATGTACAGCATC780           
ATGGCTGCTCGCTACAAGTACTTCCCGGAAGTTAAGACAAAGGGCATGGCGGCTGTGCCT840           
AAACTGGTCCTCTTCACCTCAGAACAGAGTCACTATTCCATAAAGAAAGCTGGGGCTGCA900           
CTTGGCTTTGGAACTGACAATGTGATTTTGATAAAGTGCAATGAAAGGGGGAAAATAATT960           
CCAGCTGATTTTGAGGCAAAAATTCTTGAAGCCAAACAGAAGGGATATGTTCCCTTTTAT1020          
GTCAATGCAACTGCTGGCACGACTGTTTATGGAGCTTTTGATCCGATACAAGAGATTGCA1080          
GATATATGTGAGAAATATAACCTTTGGTTGCATGTCGATGCTGCCTGGGGAGGTGGGCTG1140          
CTCATGTCCAGGAAGCACCGCCATAAACTCAACGGCATAGAAAGGGCCAACTCAGTCACC1200          
TGGAACCCTCACAAGATGATGGGCGTGCTGTTGCAGTGCTCTGCCATTCTCGTCAAGGAA1260          
AAGGGTATACTCCAAGGATGCAACCAGATGTGTGCAGGATACCTCTTCCAGCCAGACAAG1320          
CAGTATGATGTCTCCTACGACACCGGGGACAAGGCAATTCAGTGTGGCCGCCACGTGGAT1380          
ATCTTCAAGTTCTGGCTGATGTGGAAAGCAAAGGGCACAGTGGGATTTGAAAACCAGATC1440          
AACAAATGCCTGGAACTGGCTGAATACCTCTATGCCAAGATTAAAAACAGAGAAGAATTT1500          
GAGATGGTTTTCAATGGCGAGCCTGAGCACACAAACGTCTGTTTTTGGTATATTCCACAA1560          
AGCCTCAGGGGTGTGCCAGACAGCCCTCAACGACGGGAAAAGCTACACAAGGTGGCTCCA1620          
AAAATCAAAGCCCTGATGATGGAGTCAGGTACGACCATGGTTGGCTACCAGCCCCAAGGG1680          
GACAAGGCCAACTTCTTCCGGATGGTCATCTCCAACCCAGCCGCTACCCAGTCTGACATT1740          
GACTTCCTCATTGAGGAGATAGAAAGACTGGGCCAGGATCTGTAA1785                         
(2) INFORMATION FOR SEQ ID NO:15:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 29 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA                                                  
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                  
ATTGGATCCACCGAGCTGATGGCGTCTTC29                                           
(2) INFORMATION FOR SEQ ID NO:16:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 27 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA                                                  
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                  
CCGAATTCGCCATTCGCCAGCTAAACC27                                             
(2) INFORMATION FOR SEQ ID NO:17:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 28 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA                                                  
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                  
ATTGGATCCGGTTTAGCTGGCGAATGGC28                                            
(2) INFORMATION FOR SEQ ID NO:18:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 27 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA                                                  
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                  
CCGAATTCTGTGAGGGTTCCAGGTGAC27                                             
(2) INFORMATION FOR SEQ ID NO:19:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 28 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA                                                  
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                  
ATTGGATCCGTCACCTGGAACCCTCACA28                                            
(2) INFORMATION FOR SEQ ID NO:20:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 26 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA                                                  
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                  
CCGAATTCATTACAGATCCTGGCCCA26                                              
(2) INFORMATION FOR SEQ ID NO:21:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 29 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA                                                  
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                  
ACTGCCAATACCAATATGTTCACATATGA29                                           
(2) INFORMATION FOR SEQ ID NO:22:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 20 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA                                                  
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                  
CCCATAAACTCATGTTCTTG20                                                    
(2) INFORMATION FOR SEQ ID NO:23:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 20 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA                                                  
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                  
GGAGAAAATATCCCATCACC20                                                    
(2) INFORMATION FOR SEQ ID NO:24:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 28 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA                                                  
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                  
CCGAATTCTGTAGAGGGTTCCAGGTGAC28                                            
(2) INFORMATION FOR SEQ ID NO:25:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 26 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA                                                  
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                  
ATTGGATCCGGCATAGAAAGGGCCAA26                                              
__________________________________________________________________________

Claims (9)

What is claimed is:
1. A polypeptide having the amino acid sequence of SEQ ID NO:3.
2. A polypeptide consisting of the amino acid sequence of SEQ ID NO:3.
3. A polypeptide having the amino acid sequence of SEQ ID NO:4.
4. A polypeptide consisting of the amino acid sequence of SEQ ID NO:4.
5. A polypeptide having the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO:6.
6. A polypeptide consisting of the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO:6.
7. A polypeptide consisting of the polypeptide according to any one of claims 1, 3 or 5, and a contiguous second amino acid sequence.
8. A polypeptide according to claim 7 wherein said second amino acid sequence is glutathione-S-transferase (GST) or a portion thereof.
9. A polypeptide according to claim 7 wherein said second amino acid sequence comprises one or more histidine residues.
US08/308,952 1991-02-22 1994-09-20 Human islet, human brain and mouse brain glutamic acid decarboxylase GAD polypeptides Expired - Fee Related US5837812A (en)

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US09/124,141 US6211352B1 (en) 1991-02-22 1998-07-29 Method for the diagnosis and treatment of glutamic acid decarboxylase autoantigen associated diseases

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AUPK4773 1991-02-22
AUPK477391 1991-02-22
AUPK862091 1991-09-27
AUPK8620 1991-09-27
US83980592A 1992-02-21 1992-02-21
US08/308,952 US5837812A (en) 1991-02-22 1994-09-20 Human islet, human brain and mouse brain glutamic acid decarboxylase GAD polypeptides

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WO2002098891A2 (en) * 2001-06-05 2002-12-12 Exelixis, Inc. GADs AS MODIFIERS OF THE p53 PATHWAY AND METHODS OF USE
US20030027246A1 (en) * 2000-03-31 2003-02-06 Artur Pedyczak Immunogenic peptides derived from prostate-specific membrane antigen (PSMA) and uses thereof
US20040009906A1 (en) * 2002-05-06 2004-01-15 Kakkis Emil D. Induction of antigen specific immunologic tolerance
US20050191309A1 (en) * 2002-05-06 2005-09-01 Kakkis Emil D. Induction of antigen specific immunologic tolerance
US7361331B2 (en) 1996-10-18 2008-04-22 Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of Agriculture And Agri-Food Plant bioreactors

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US5998366A (en) * 1990-09-21 1999-12-07 The Regents Of The University Of California Method for ameliorating glutamic acid decarboxylase associated autoimmune disorders
US6682906B1 (en) * 1990-09-21 2004-01-27 The Regents Of The University Of California Cloned glutamic acid decarboxylase
WO1992020811A2 (en) * 1991-05-15 1992-11-26 Zymogenetics, Inc. Cloning and expression of human islet glutamic acid decarboxylase autoantigen
AU661684B2 (en) * 1991-06-18 1995-08-03 Regents Of The University Of California, The Cloned glutamic acid decarboxylase
DE4237244A1 (en) * 1992-11-04 1994-05-05 Elias Entwicklungslabor Fuer I Process for the production of high-purity human GAD-1 and GAD-2 proteins
AU688304B2 (en) * 1993-02-09 1998-03-12 Monash University Methods for the diagnosis of diabetes and prediabetic conditions
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EP1849870A1 (en) 1995-02-20 2007-10-31 The Walter And Eliza Hall Institute Of Medical Research Immunoreactive and immunotherapeutic molecules which interact in subjects with insulin-dependent diabetes mellitus (IDDM)
JP2000500120A (en) * 1995-09-21 2000-01-11 ユニバーシティ・オブ・ユタ・リサーチ・ファウンデーション Targeting of conjugate of poly (ethylene glycol) and anti-glutamate decarboxylase antibody to pancreatic islet cells
EP0923944A4 (en) * 1996-04-01 2000-05-03 Chugai Pharmaceutical Co Ltd Diagnostic composition for diseases accompanied by autoimmune reactions caused by 65k-glutamic acid decarboxylase
US7189516B2 (en) * 2001-08-17 2007-03-13 Luminex Corporation Method for characterizing autoimmune disorders
WO2006130639A2 (en) * 2005-05-31 2006-12-07 Neurologix, Inc. Novel glutamic acid decarboxylase (gad) chimera and methods of use

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US7361331B2 (en) 1996-10-18 2008-04-22 Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of Agriculture And Agri-Food Plant bioreactors
US20030027246A1 (en) * 2000-03-31 2003-02-06 Artur Pedyczak Immunogenic peptides derived from prostate-specific membrane antigen (PSMA) and uses thereof
WO2002098891A2 (en) * 2001-06-05 2002-12-12 Exelixis, Inc. GADs AS MODIFIERS OF THE p53 PATHWAY AND METHODS OF USE
WO2002098891A3 (en) * 2001-06-05 2003-05-30 Exelixis Inc GADs AS MODIFIERS OF THE p53 PATHWAY AND METHODS OF USE
US20040009906A1 (en) * 2002-05-06 2004-01-15 Kakkis Emil D. Induction of antigen specific immunologic tolerance
US20050191309A1 (en) * 2002-05-06 2005-09-01 Kakkis Emil D. Induction of antigen specific immunologic tolerance
US7485314B2 (en) 2002-05-06 2009-02-03 Los Angeles Biomedical Research Institute At Harbor-Ucla Medical Center Induction of antigen specific immunologic tolerance
US20090238818A1 (en) * 2002-05-06 2009-09-24 Biomarin Pharmaceutical Inc. Induction of Antigen Specific Immunnologic Tolerance
US7883707B2 (en) 2002-05-06 2011-02-08 Kakkis Emil D Induction of antigen specific immunnologic tolerance

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CA2104225A1 (en) 1992-08-23
WO1992014485A1 (en) 1992-09-03
EP0572478B1 (en) 2005-09-07
DE69233507D1 (en) 2005-06-16
ATE304050T1 (en) 2005-09-15
JP3027190B2 (en) 2000-03-27
DE69233507T2 (en) 2009-09-10
EP0572478A1 (en) 1993-12-08
AU1274892A (en) 1992-09-15
EP0572478A4 (en) 1994-05-18
JPH06505385A (en) 1994-06-23
CA2104225C (en) 2001-07-17
US6211352B1 (en) 2001-04-03
AU659133B2 (en) 1995-05-11

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